To date our genetic and biochemical studies indicate that our original Hsp70 mutant impairs prions either by promoting self-association of amyloid fibers into large aggregates or by interfering with the disentangling of amyloid fibers from such higher-order aggregates. Unlike other in vitro systems used for studying interaction of chaperones with amyloid, the system we are developing allows testing the ability of chaperones or chaotropic agents to disentangle aggregated amyloid fibers. The basis of the system is a version of a multiply tagged yeast prion domain that can be differentially labeled by fluorescent and other moieties. Fibers can be labeled by either or both probes, and those containing both can be labeled either sequentially or simultaneously. Fluorescence can be used in conjunction with physical methods such as fractionation and immunoblotting to test the extent to which chaperones break fibers. Our system will also allow monitoring the action of chaperones in the disentangling of polymers from aggregates. Ultimately, products generated from in vitro reactions will be used for in vivo experiments to test our predictions of how chaperone interaction with amyloid affects prion infectivity.[unreadable] At present the system is incomplete but showing promise. We believe the rewards of establishing such a system for uncovering molecular mechanisms to explain a large amount of genetic data obtained for both wild type and mutant chaperones merit continued development. To date no laboratory has shown effects of any mutant chaperone or co-chaperone on amyloid in an in vitro system. Insight gained from our new system should allow us to correlate effects our mutant chaperones have on amyloid with effects they have on prion phenotype and thus contribute considerably to our understanding of amyloid propagation in vivo.